In humans, the spleen serves at least four major physiologic functions. First, as part of the peripheral immune system, it clears the blood of microorganisms and particulate antigens and/or generates antigens to foreign substances. Second, it sequesters and removes excess, old and/or abnormal blood cells. Third, its vasculature is involved in the regulation of portal blood flow. Finally, it engages in hematopoiesis during development or when the bone marrow alone cannot produce sufficient blood cells.
The spleen consists of red pulp which contains blood-filled sinuses and pulp cords lined by reticuloendothelial cells and of white pulp which is arranged around a central arteriole. The surrounding periarteriolar lymphoid sheath (PALS) contains both T and B cell areas. The T cell area lies adjacent to the arteriole and consists of small, densely packed lymphocytes, primarily T4.sup.+ helper T lymphocytes. Outside of the T cell area is the follicular zone which contains B lymphocytes and germinal centers which are made up of B cells and macrophages. The white pulp is surrounded by a marginal zone containing specialized, antigen-presenting macrophages and B cells.
Blood-borne lymphocytes enter the red and white pulp via the trabecular artery. Most branches empty into or near the marginal zones; but some end in the white pulp and supply oxygen and nutrients to the germinal centers and outer mantle zones, and others run directly into the red pulp. T and B lymphocytes pass through the spleen at different rates. B cells generally migrate through the tissue more slowly, but they eventually cross into the red pulp and exit the spleen via the splenic vein.
In the spleen as well as other tissues, leukocytes including monocytes, macrophages, basophils, and eosinophils play important roles in the pathological mechanisms initiated by T and/or B lymphocytes. Macrophages, in particular, produce powerful oxidants and proteases which contribute to tissue destruction and secrete a range of cytokines which recruit and activate other inflammatory cells.
The investigation of the critical, regulatory processes by which white cells proceed to their appropriate destination and interact with other cells is underway. The current model of leukocyte movement or trafficking from the blood to injured or inflamed tissues comprises the following steps. The first step is the rolling adhesion of the leukocyte along the endothelial cells of the blood vessel wall.. This movement is mediated by transient interactions between selectins and their ligands. A second step involves cell activation which promotes a more stable leukocyte-endothelial cell interaction mediated by the integrins and their ligands. This stronger, more stable adhesion precipitates the final steps--leukocyte diapedesis and extravasation into the tissues.
The chemokine family of polypeptide cytokines possesses the cellular specificity required to explain leukocyte trafficking in different abnormal, inflammatory or diseased situations. First, chemokines mediate the expression of particular adhesion molecules on endothelial cells; and second, they generate gradients of chemoattractant factors which activate specific cell types. In addition, the chemokines stimulate the proliferation of specific cell types and regulate the activation of cells which bear specific receptors. These activities demonstrate a high degree of target cell specificity.
The chemokines are small polypeptides, generally about 70-100 amino acids (aa) in length, 8-11 kD in molecular weight and active over a 1-100 ng/ml concentration range. Initially, they were isolated and purified from inflamed tissues and characterized relative to their bioactivity. More recently, chemokines have been discovered through molecular cloning techniques and characterized by structural as well as functional analysis.
The chemokines are related through a four-cysteine motif which is based primarily on the spacing of the first two cysteine residues in the mature molecule. Currently the chemokines are assigned to one of two families, the C-C chemokines (.alpha.) and the C-X-C chemokines (.beta.). Although exceptions exist, the C-X-C chemokines generally activate neutrophils and fibroblasts while the C-C chemokines act on a more diverse group of target cells which include monocytes/macrophages, basophils, eosinophils, T lymphocytes and others. The known chemokines of both families are synthesized by many diverse cell types as reviewed in Thomson A. (1994) The Cytokine Handbook, 2d Ed. Academic Press, New York. The two groups of chemokines will be described in turn.
At this time, relatively few C-C chemokines have been described, and they appear to have less N-terminal processing than the C-X-C chemokines. A brief description of the known human (and/or murine) C-C chemokines follows. The macrophage inflammatory proteins alpha and beta (MIP-1.alpha.and .beta.) were first purified from stimulated mouse macrophage cell line and elicited an inflammatory response when injected into normal tissues. At least three distinct and non-allelic genes encode human MIP-1.alpha., and seven distinct genes encode MIP-1.beta..
MIP-1.alpha.and MIP-1.beta.consist of 68-69 aa which are about 70% identical in their acidic, mature secreted forms. They are both expressed in stimulated T cells, B cells and monocytes in response to mitogens, anti-CD3 and endotoxin, and both polypeptides bind heparin. While both molecules stimulate monocytes, MIP-1.alpha.chemoattracts the CD-8 subset of T lymphocytes and eosinophils, while MIP-1.beta.chemoattracts the CD-4 subset of T lymphocytes. In mouse, these proteins are known to stimulate myelopoiesis.
I-309 was cloned from a human .gamma.-.delta.T cell line and shows 42% aa identity to T cell activation gene 3 (TCA3) cloned from mouse. There is considerable nucleotide homology between the 5' flanking regions of these two proteins, and they share an extra pair of cysteine residues not found in other chemokines. Such similarities suggest I-309 and TCA3 are species homologs which have diverged over time in both sequence and function.
RANTES is another C-C chemokine which is expressed in T cells (but not B cells), in platelets, in some tumor cell lines, and in stimulated rheumatoid synovial fibroblasts. In the latter, it is regulated by interleukins-1 and -4, transforming nerve factor and interferon-.gamma.. The cDNA cloned from T cells encodes a basic 8 kD protein which lacks N-linked glycosylation and is able to affect lymphocytes, monocytes, basophils and eosinophils. The expression of RANTES mRNA is substantially reduced following T cell stimulation.
Monocyte chemotactic protein (MCP-1) is a 76 aa protein which appears to be expressed in almost all cells and tissues upon stimulation by a variety of agents. The targets of MCP-1, however, are limited to monocytes and basophils in which it induces a MCP-1 receptor:G protein-linked calcium flux (Charo I, personal communication). Two other related proteins (MCP-2and MCP-3) were purified from a human osteosarcoma cell line. MCP-2 and MCP-3 have 62% and 73% aa identity, respectively, with MCP-1 and share its chemoattractant specificity for monocytes.
Current techniques for diagnosis of abnormalities in the inflamed or diseased tissues mainly rely on observation of clinical symptoms or serological analyses of body tissues or fluids for hormones, polypeptides or various metabolites. Patients often manifest no clinical symptoms at early stages of disease or tumor development. Furthermore, serological analyses do not always differentiate between invasive diseases and genetic syndromes which have overlapping or very similar ranges. Thus, development of new diagnostic techniques comprising small molecules such as the expressed chemokines are important to provide for early and accurate diagnoses, to give a better understanding of molecular pathogenesis, and to use in the development of effective therapies.
The chemokine molecules were reviewed in Schall TJ (1994) Chemotactic Cytokines: Targets for Therapeutic Development. International Business Communications, Southborough, Mass., pp 180-270; and in Paul WE (1993) Fundamental Immunology, Raven Press, New York City (NYC), pp 822-826.